1. Field of the Invention
The present invention relates to a novel member of the NK family of homeobox genes. More specifically, isolated nucleic acid molecules are provided encoding a human NK-3 related prostate specific gene (NKX3.1). NKX3.1 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of NKX3.1 activity. Also provided are diagnostic methods for detecting prostate cancer and other cancers and therapeutic methods for prostate cancer and other cancers.
2. Related Art
The discovery of the homeobox as a conserved DNA sequence element in several Drosophila genes responsible for controlling the identity of body segments prompted searches for related genes in other organisms. Homeoboxes have since been discovered in the genome of all metazoan organisms, and several hundred unique homeobox genes have been defined in mice and humans (Gehring, W. J. et al., Annu. Rev. Biochem. 63:487-526 (1994); Stein, S. et al., Mech. Develop. 55:91-108 (1996)). The homeobox encodes a 60-amino acid domain, termed the homeodomain, that includes a helix-turn-helix motif recognized to be structurally-related to the DNA binding domain of several procaryotic proteins and to the products of the yeast mating type locus (Laughon, A. and Scott, M. P., Nature 310:25-31 (1984); Shepherd, J. C. W. et al., Nature 310:70-71 (1984)). NMR and crystallographic analyses have confirmed that the homeodomain binds DNA (Kissinger, C. R. et al., Cell 63:579-590 (1990); Otting, G. et al., EMBO J. 9:3085-3092 (1990)). As predicted by the nature of the phenotypes produced when these genes are mutated, both biochemical and genetic analyses have established that the products of homeobox genes are transcriptional regulatory molecules (McGinnis, W. and Krumlauf, R., Cell 68:283-302 (1992)).
The predicted amino acid sequence of the known homeodomains serves as the principal identifier that allows them to be classified into a minimum of 20 distinct groups (Gehring, W. J. et al., Annu. Rev. Biochem. 63:487-526 (1994); Stein, S. et al., Mech. Develop. 55:91-108 (1996)). The NK family of homeobox genes, first defined by four related Drosophila genes, NK-1 through NK-4, can be separated into two distinct classes. NK-2, -3 and -4 are more related to each other than to other homeobox genes, whereas NK-1 is a more distant relative (Kim, Y. and Nirenberg, M., Proc. Natl. Acad. Sci. USA 86:7716-7720 (1989)). In mouse, six NK-2-like genes have been identified (Price, M. et al., Neuron 8:241-255 (1992); Lints, T. J. et al., Development 119:419-431 (1993)). Three of these are more related to NK-2 than the others, which may themselves form a distinct subclass (Lints, T. J. et al., Development 119:419-431 (1993)).
The majority of studies aimed at characterizing the functions of homeobox genes have focused principally on their developmental roles (McGinnis, W. and Krumlauf, R., Cell 68:283-302 91992); Krumlauf, R., Cell 78:191-201 (1994)). A prominent example is the Hox family of genes, whose members have been demonstrated to play critical roles in pattern formation during embryogenesis along the anteroposterior body axis of divergent species (Krumlauf, R., Cell 78:191-201 (1994)). Some of the Hox genes, as well as members of other classes of homeobox genes, are also expressed during organogenesis, and a few of these have been reported to be expressed in adult tissues. Surprisingly, the potential roles of homeobox genes in fully differentiated tissues and organs have received comparatively little attention. However, the need for patterning functions to maintain the differentiated states of cell populations and to direct the renewal of specific cell types in adults is axiomatic.
The mechanisms involved in the development and maintenance of prostatic tissue are poorly understood. Although it has been recognized for years that normal development and continued expression in adults of the male secondary sexual phenotype is androgen-dependent, there is relatively little known about the genes on which androgens act or the downstream pathways that lead to development of differentiated tissue. As with prostate development, the fundamental mechanisms underlying prostate cancer also remain obscure, however, androgen regulation and the loss thereof plays a critical role. In both developing and mature prostate, the maintenance of prostate-specific cellular functions requires continuous stimulation by androgens; in prostate cancer tissue, the reciprocal loss of this cellular differentiation, which occurs during progression of the disease, is largely concomitant with a loss of androgen responsiveness by prostatic cells. Identifying the genes involved in either of these largely opposing process, will likely lead to a greater understanding of the fundamental mechanisms involved in both.
Thus far, no genes are known to play a key role in the progressive loss of differentiated phenotype seen in prostate cancer tissue, but various studies indicate the presence of one or more genes on human chromosome 8p that suppress the occurrence and/or progression of the disease. Several investigators have found, based on loss of heterozygosity (LOH) studies, that chromosome bands 8p21 contain loci that are deleted in up to 80% of prostate cancer tissues (Suzuki, et al., Genes, Chromosomes and Cancer 13:168-174 (1995), Bova et al., Cancer Res. 53:3869-3873 (1993), MacGrogan et al., Genes, Chromosomes and Cancer 10:151-159 (1994), Trapman et al., Cancer Res. 54:6061-6064 (1994), Macoska, et al., Cancer Res. 55:5390-5395 (1995), and Vocke et al., Cancer Res. 56:2411-2416 (1996)). In addition, the introduction of human chromosome 8 into the highly metastatic Dunning rat prostate cancer cell line significantly reduces its metastatic potential (Ichikawa et al., Cancer Res. 54:2299-2302 (1994)). The loss of 8p during the derivation of subclones from the human prostate cancer line, LNCaP, is correlated with loss of androgen responsiveness (Konig et al., Urol. Res. 17:79-86 (1989)).
A prostate-specific human gene (NKX3.1) which maps to 8p21 and encodes a homeodomain-containing protein related to the Drosophila NK gene family was cloned. The gene may play a role in both prostate development and the androgen-driven maintenance of prostatic differentiation in adults. The expression of NKX3.1 in adult humans is restricted to prostate and testes and when assayed in several cell lines, including three lines derived from prostate carcinoma tissue, the gene was expressed solely in the androgen-dependent prostate carcinoma cell line LNCaP. A detailed study of NKX3.1 expression in LNCaP cells has demonstrated that the gene is transcriptionally regulated by androgens. Thus, the new prostate-specific gene NKX3.1 is a candidate for playing a central role in the opposing processes of androgen-driven differentiation of prostatic tissue and loss of that differentiation during the progression of prostate cancer.
Thus, the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the human NK-3 related prostate specific gene 1 (NKX3.1) polypeptide having the amino acid sequence shown in FIG. 1 or 2 (SEQ ID NOs:2 or 4) or the amino acid sequence encoded by the cDNA clone deposited as ATCC Deposit Number 209006 on Apr. 28, 1997. The present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding the human NK-3 related prostate specific gene 1 (HPFCA19) polypeptide having the amino acid sequence shown in FIG. 5 (SEQ ID NO:9) or the amino acid sequence encoded by the genomic clone deposited as ATCC Deposit Number 209005 on Apr. 28, 1997.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of NKX3.1 polypeptides or peptides by recombinant techniques.
The invention further provides an isolated NKX3.1 polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by the NKX3.1, which involves contacting cells which express the NKX3.1 with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
The invention provides a diagnostic method useful for diagnosis of prostate cancer and other cancers.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of NKX3.1 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated NKX3.1 polypeptide of the invention or an agonist thereof.